An automatic nucleic acid purification apparatus is mainly used to isolate nucleic acid from a biological sample. This is caused by that a quantitative test can be high-sensitively and precisely performed with respect to target pathogenic bacteria of from 10 or less to one billion using real-time quantitative PCR. However, the nucleic acid purification is simultaneously performed with respect to multiple biological samples. Therefore, in case that the purification is simultaneously performed with respect to a biological sample containing high concentration target pathogenic bacteria and another biological sample not containing target nucleic acid, a false positive may be occurred by pollution due to aerosol. For example, in case of purifying target nucleic acids in a biological sample containing one billion viruses per 1 ml, even when another biological sample is polluted with a hundred millionth ml of the aerosol generated from the biological sample, i.e., 1 pl (picoliter) of aerosol, the false positive may be occurred.
Generally, a material to which nucleic acid is selectively attached is used in purifying nucleic acid. There are a method using a membrane and a method using magnetic particles. Typically, the method using magnetic particles is more widely used. In this method, instead of a vacuum manner in which it is easy to generate the aerosol, biochemistry substance is rapidly attached to magnetic particles suspended in a solution, and the magnetic particles to which the target substance is attached is cohered by magnetic field, and then the solution is removed. There has been developed various relevant automatic equipments.
According to an attaching manner of the magnetic particles, this is classified into a method of attaching the magnetic particles to a pipette, a method of attaching the magnetic particles to a tube in which a magnet rod is inserted, and a method of the magnetic particles to a multi-well plate.
Recently, an automatic method using a pipette is wisely used. In U.S. Pat. No. 5,647,994 (Labsystems Co., Ltd.), there have been described various methods of separating magnetic particles using a disposable pipette. Also, in U.S. Pat. Nos. 5,702,950 and 6,231,814 (Precision System Science Co., Ltd.), magnetic particles are attached to a pipette, and a basic principle thereof is the same as in U.S. Pat. No. 5,647,994. A difference between them is that a magnet is attached and detached in one direction of the pipette and thus magnetic field is controlled in one direction of a pipette tip. This patent is characterized in that a magnetic substance attracting/releasing control method comprises the steps of: providing a pipette device having a liquid suction line including a liquid inlet end for sucking liquid containing the magnetic substance from a container and discharging the liquid through the liquid inlet end, and a magnet body or magnet bodies being detachably fitted to an external peripheral surface of the liquid suction line of the pipette device; the pipette device providing attracting/releasing control by absorbing and maintaining the magnetic substance contained in the liquid and attracted to the liquid suction line due to magnetism in the magnet body or bodies on an internal surface of the liquid suction line, the magnetic substance being maintained on the internal surface of the pipette device and also by releasing the magnetic substance from the liquid suction line by means of interrupting effect by magnetism in the magnet body or bodies so that the substance is discharged together with the liquid to outside of the liquid suction line through the liquid inlet end.
In U.S. Pat. No. 6,187,2070 (Roche Diagnostics GmbH), there is disclosed a method of separation of magnetic particles, in which a permanent magnet is approached to a disposable tip so as to adhere the magnetic particles, thereby separating the magnetic particles from a solution. To this end, an apparatus for separation of the magnetic particles includes a pipette connected to a pump, a magnet, and a means for moving the magnet to the pipette side or the opposite side thereof. Herein, there is provided the method of separating the magnetic particles from the solution and then suspending them in another solution. However, one of the uppermost limits is that a lower portion of the pipette is clogged with the magnetic particles, and thus the results become inaccurate.
There has been proposed a method for nucleic acid purification, in which magnetic particles are attached to a detachable magnet rod and then moved to various solutions for nucleic acid purification. To this end, there has been developed various models such as Maxwell 16™ manufactured by Promega Co., Ltd and King Fisher™ manufactured by Thermo Co., Ltd. In these systems, since the solution is stirred by moving up and down a tube in which a rod for collecting the magnetic particles is inserted, the entire part of the tube is smeared with the solution containing nucleic acid.
In a conventional method, a reaction is performed in a container in which a biological sample is received, and magnetic particles are attached to a desired place, and then the purification is carried out. By Gen-Probe, Inc., there has been proposed a method for separating particles, which are attracted by magnetic field, using a separation rack. In U.S. Pat. No. 5,897,783 (Amersham International plc), there has been disclosed a method of moving a doughnut-shaped permanent magnet in a vertical direction to a container and thus switching magnetic field. In EP0479448 (Beckman instruments, Inc), there has been disclosed an automatic purification apparatus in which a sample containing magnetic materials is separated using a magnetic plate. The magnetic plate is formed with a plurality of holes through which a container can be passed, and the automatic purification apparatus also includes a means for moving up and down the magnetic plate. In case that magnetic particles are attached to a bottom portion, mixing of a magnetic particle suspension, a sample and a solution is occurred in a dispensable pipette, and thus the solution containing nucleic acid is smeared on an outer surface of the pipette.
When performing purification of biological substances, all of the above-mentioned methods essentially include a step of moving a pipette or a tube, on which the solution containing nucleic acid is smeared due to binding of Lysis and magnetic particles, a cleaning reaction and the like, to another space. In this step, since a vortex of air is generated on the outer surface of the dispensable pipette or the purification tube, pollution due to aerosol is unavoidable.
However, in the automatic purification apparatuses which have been developed till now, consideration for efficiently preventing the generation of aerosol is yet insignificant. As a pollution preventing system which has been developed till now, a solution drip guard for preventing solution drips from a dispensable pipette is applied to Exiprep 16 Pro manufactured by Bioneer Corporation and to MagnaPure 96 manufactured by Roche. But even though it is possible to prevent the solution drips from the pipette, it is not possible to prevent the aerosol generated from the solution containing nucleic acid attached to the hydrophobic surface of the pipette by a vortex of air generated when the pipette is moved. Therefore, it is inevitable that the aerosol is generated during a series of processes for separating target nucleic acid from a biological sample solution, and particularly, it is not possible to avoid false positive pollution in PCR due to the aerosol generated from the solution containing high concentration nucleic acid.
The present invention is to minimize the generation of aerosol generated from fine drips attached on the outer surface of the pipette by air flow and thus to minimize a cross pollution.